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Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Clinical Medicine College of Guizhou Medical University, Guiyang, ChinaDepartment of Hematology, Affiliated Hospital of Guizhou Medical University, Guizhou Province Institute of Hematology, Guizhou Province Laboratory of Hematopoietic Stem Cell Transplantation Centre, Guiyang, China
Recently, immune escape has been considered as a factor leading to relapse of acute myeloid leukemia (AML). In our previous study, heme oxygenase 1 (HO-1) proved to play an essential role in the proliferation and drug resistance of AML cells. In addition, recent studies by our group have shown that HO-1 is involved in immune escape in AML. Nevertheless, the specific mechanism by which HO-1 mediates immune escape in AML remains unclear.
Methods
In this study, we found that patients with AML and an overexpression of HO-1 had a high rate of recurrence. In vitro, overexpression of HO-1 attenuated the toxicity of natural killer (NK) cells to AML cells. Further study indicated that HO-1 overexpression inhibited human leukocyte antigen-C and reduced the cytotoxicity of NK cells to AML cells, leading to AML relapse. Mechanistically, HO-1 inhibited human leukocyte antigen-C expression by activating the JNK/C-Jun signaling pathway.
Results
In AML, HO-1 inhibits cytotoxicity of NK cells by inhibiting the expression of HLA-C, thus causing immune escape of AML cells.
Conclusions
NK cell-mediated innate immunity is important for the fight against tumors, especially when acquired immunity is depleted and dysfunctional, and the HO-1/HLA-C axis can induce functional changes in NK cells in AML. Anti-HO-1 treatment can promote the antitumor effect of NK cells and may play an important role in the treatment of AML.
Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells with abnormal differentiation of myeloid primordial naive cells, which has the greatest incidence in acute adult leukemia, with a low cure rate [
]. Clinically, less than one-third of adult patients with AML can achieve sustained remission after cytarabine combined with anthracycline chemotherapy, and more than 50% is detected after remission [
Novel agents targeting leukemia cells and immune microenvironment for prevention and treatment of relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation.
]. Allogeneic hematopoietic stem cell transplantation is still the only way to cure leukemia clinically. However, because of age limitations, infection, graft-versus-host disease, and other complications, it is not suitable for all patients with AML [
]. Recently, numerous novel drugs have provided more options for treating relapsed and refractory AML. At the same time, the overall response rate is unsatisfactory, and there is no significant advantage in improving overall survival [
]. Therefore, exploring the mechanism affecting AML relapse and refractory treatment and formulating effective coping strategies are the keys to overcoming the treatment bottleneck of AML.
Heme oxygenase 1 (HO-1) is one of the essential oxidation regulatory enzymes of the body. Its primary physiological function is to degrade oxidized heme into free iron oxide, carbon monoxide (CO), and biliverdin [
], which can be quickly converted to bilirubin, protect mammalian cells, is involved in the maintenance of steady state, and can reduce oxidative damage and inflammatory response [
HO-1 promotes resistance to an EZH2 inhibitor through the pRB-E2F pathway: Correlation with the progression of myelodysplastic syndrome into acute myeloid leukemia.
Natural killer (NK) cells belong to a group of large granular lymphocytes, which can be an essential component of innate immunity. Greater tumor susceptibility and metastasis have been associated with the reduced cytotoxicity of NK cells in mouse models and clinical studies [
]. NK cells can rapidly and nonspecifically identify target cells to exert the anti-AML effects, which is not limited by major histocompatibility antigens (MHCs) [
]. The cytotoxicity of NK cells mainly depends on the signal transduction of its surface receptors. Activation receptors expressed on the surface of NK cells are the natural cytotoxic receptors, including NKp30, NKp46, NKG2D and part of the killer-cell immunoglobulin-like receptor family. They can bind to human leukocyte antigen-I (HLA-I) molecules (HLA-A/B/C/D/E/G/Hs) to activate the effect of activating NK cells. The increased expression of HLA-I molecules in AML cells can lead to the activation of the already-inhibited NK cells and exert a killing effect [
Natural killer cells in myeloid malignancies: Immune surveillance, NK cell dysfunction, and pharmacological opportunities to bolster the endogenous NK cells.
]. However, whether HO-1 mediates the immune escape of AML cells through HLA-C is still unclear.
Methods
Patient specimens and cell lines
Bone marrow samples were collected from 30 patients with AML at The Affiliated Hospital of Guizhou Medical University from September 2020 to May 2021 using a simple random sampling method. Detailed clinical data are shown in supplementary Table 1. Before treatment, patient samples were collected at diagnosis and relapse. We previously obtained patient consent and approval from the institutional research Ethics Committee.
Human AML cell lines U937 and THP-1 were obtained from the laboratory of Guizhou Hematopoietic Stem Cell Transplantation Center. All human cell lines were tested for mycoplasma contamination and validated by a short tandem repeat map. Cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 mg/mL) at 37°C and 5% CO2.
Reagents and antibodies
Hemin, Znpp and SP600125 (JNK inhibitors) were purchased from MCE (Taiwan, China). Fetal bovine serum and RPMI 1640 medium were obtained from Gibco (Carlsbad, CA, USA). Signaling Technology (Danvers, MA, USA) supplied anti-JNK (#9252), anti-phosphorylated JNK (#4668), anti-phosphorylated c-Jun (#2361) and anti-c-Jun (#9165) antibodies for western blot analysis. Anti-HO-1 (K009440M), anti-β-actin (K101527P) and anti-HLA-C (K001854P) antibodies were obtained from Solarbio (Beijing, China) for western blotting. Anti-HLA-C (566372) antibody was obtained from BD Biosciences (San Jose, CA, USA) for flow cytometry. Anti-HO-1 (K009440M) and anti-HLA-C (K001854P) were obtained from Solarbio for immunocytochemistry (ICC) and immunohistochemistry (IHC).
RNA extraction and semi-quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs from cells were extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RT-PCR was performed using the SYBR Green PCR Master Mix (TianGen Biotech, Beijing, China) and the PRISM 7500 RT-PCR detection system (ABI, San Diego, USA). The following primers were synthesized by Transheep Biotechnology Co. Ltd: HO-1, forward 5′-ACCCATGACACCAAGGACCAGA-3′ and reverse 5′-GTGTAAGGACCCATCGGAGAAGC-3′; HLA-C, forward 5′-CTGCCTGTGTGGGACTGA-3′ and reverse 5′-GGGGTGGGCTGTCTCTC-3′; β-actin, forward 5′-GAGACCTTCAACACCCCAGC-3′ and reverse 5′-ATGTCACGCACGATTTCCC-3′. Each sample was repeated twice, and each cell experiment was repeated at least three times. Results were analyzed using the PRISM 7500 real-time PCR detection system (ABI, San Diego, USA). The fold change was determined using the ΔΔCt method. Gene expression was normalized using β-actin RNA.
Western blot analysis
Cells were collected and extracted with RIPA lysis buffer (50 mol/L Tris HCl, 150 mol/L NaCl, 5 mol/L ethylenediaminetetraacetic acid, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl-sulfate, 1% aprotinin, 50 mol/L NaF and 0.1 mol/L Na3VO4) to detect changes in cellular protein levels. Samples with equal amounts of protein were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene fluoride membranes (Millipore Corporation, Burlington, MA, USA) and then incubated with the corresponding primary antibodies for HO-1 (1:1000), HLA-C (1:1000), p-JNK (1:1000), JNK (1:1000), p-c-Jun (1:1000), c-Jun (1:1000) or β-actin (1:1000) overnight at 4°C after blocking with 5% nonfat milk for 2 hours. The membranes were washed and then incubated with horseradish peroxidase–conjugated secondary antibody (1:2000) for 1 hour at room temperature. The signal was determined by enhanced chemiluminescence (7 Sea Biotech, Shanghai, China). The expression levels of related proteins were semi-quantified and normalized against β-actin using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Lentiviral transduction
Human HO-1 overexpressed and silenced cloned Lentivirus particles were purchased from Genechem Chemical Co., Ltd. (Shanghai, China). The transfection of HO-1 was carried out according to the manufacturer's instructions. THP-1 and U937 cells were transfected with a blank vector (EV) as control. THP-1 and U937 stable cell lines expressing L-HO-1 or Si-HO-1 were screened by puromycin (1.5 μg/mL and 2 μg/mL, respectively) after amplification and maintenance in RPMI-1640 medium supplemented with 10% fetal bovine serum for 5 days.
Small interfering RNA (siRNA) transduction
HLA-C for siRNA was purchased from Shanghai Chuanyang Biological Co., Ltd. Four HLA-specific small infection sequences were co-cultured with THP-1 cell line and HO-1 down-regulated U937 cell line for 24 h. Western Blot was used to detect the stable transfection of small interference sequences. U937 and THP-1 were co-cultured with stably transfected siRNA for 24 h; Namipo was used to improve the infection efficiency.
Cytotoxicity and degranulation assays
Blood samples from different healthy donors were extracted, and peripheral blood mononuclear cells (PBMCs) were extracted within 1 h. The information for NK cells in PBMCs was detected by flow cytometry. PBMCs were co-cultured with U937 and THP-1 at an effect target ratio of 10:1. PE/Cyanine7 Anti-human CD107a Antibody (BioLegend, San Diego, CA, USA), Monensin and bredinin (BioLegend) were co-cultured for 6 h at 37°C and 5% CO2. CD56 antibody was added after 6 h of co-culture, then CD107a and CD56 were detected by flow cytometry (BD Biosciences). The experiment was repeated three times with different healthy donors.
The NK-cell–associated cytotoxicity was explored by the Cytotoxicity Detection Kit PLUS (Roche, Reinach, Switzerland) and was based on the determination of lactate dehydrogenase (LDH) from damaged cells. Purified NK cells (as effector cells) were incubated with transduced THP-1 or MV4-11 cells (as target cells) at various effector cell/target cell ratios (2:1,4:1, 6:1, 8:1, 10:1) in 96-well plates for 3.5 hours. Test samples were prepared in triplicates. Reaction mixtures, as well as the stop solution, were sequentially added to each well. Sample absorbance was determined at 490 nm using an enzyme-linked immunosorbent assay reader. The levels (%) of NK-cell–associated cytotoxicity were determined based on the ODs as Cytotoxicity (%) = (effector/target cell mix-effector cell control-low control)/(high control-low control) × 100.
Cytokine assay
Venous blood was collected from patients with AML, coagulated naturally at room temperature for at least 30 min and centrifuged at 1000g for 10 min. The isolated serum was used to detect cytokine expression on flow cytometry using a cytokine detection kit (multi-sphere flow immunofluorescence luminescence; Raisecare, Qingdao, China).
ICC and IHC staining
ICC staining requires AML mononuclear cells to be fixed in formaldehyde for 30 min. After being washed with phosphate-buffered saline (PBS) three times, the cells were then immersed in PBS containing 0.1% Triton-X 100 for 20 min. After 5 min of repair, they were sealed with 5% bovine serum albumin for 1 h at room temperature. They were then diluted (1:100 rabbit anti-HLA-C; 1:500 rabbit anti-HO-1) and treated overnight at 4°C. The cells were incubated with horseradish peroxidase second antibody (1:200) for 1 h and then washed three times with PBS. The cells were incubated for 10 min, rinsed with PBS for 10 min and then stained with hematoxylin for 1 min. They were dehydrated with gradient ethanol, transparent with xylene for 5 min, and finally photographed under a microscope. IHC assays were graded as described in Guo et al. [
Enriched NK cells were isolated from healthy donor PBMCs using the NK cell culture kit (T&L Biological Technology; cat. no: AS-01) in vitro. The enriched NK cells are used for mouse tail vein injection and LDH experiment. All experiments were performed based according to the principles of the Declaration of Helsinki. Informed consent (written) was acquired from all donors before blood collection, whereas ethical approval was granted by the Guizhou Medical University.
Xenografted tumor model
Nonobese diabetic/severe combined immunodeficiency mice were purchased from the Sibefu Biotechnology Co., Ltd (Beijing, China). Stable transfected HO-1 cells were resuspended in PBS at a concentration of 5 × 106 cells/100 μL and then injected subcutaneously into 5-week-old male mice. The mice were randomly divided into an EV group, Si HO-1 group, EV + NK group, and Si HO-1 + NK group (n = 3 in each group). When the tumor was visible or palpable, the mice were injected with enriched NK cells (1 × 106 cells/2 days) through the tail vein until death. Tumor weight and diameter were measured weekly. All mouse experiments were approved by the Animal Care and Use Committee of Guizhou Medical University. Although this experiment is not blind, it avoids introducing bias in the evaluation of experimental data.
Statistical analysis
Data are presented as the mean ± standard deviation. Comparisons between two groups were performed with Student's t-test or non-parametric Mann–Whitney U test when appropriate. One-way analysis of variance was used to estimate differences between three or more groups. A Pearman correlation analysis was used to assess the strength of association between HO-1 mRNA level and HLA-C mRNA level in patients with AML. P values less than 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA).
Results
The high recurrence rate and dysfunction of NK cells in patients with AML with high HO-1 expression cancer cells
Increasing evidence has shown that HO-1 plays an important role in tumor cell proliferation and immune escape [
]. In our study, specimens from patients with AML were divided into two groups with high or low HO-1 expression level according to qRT-PCR results, with the median HO-1 expression level being taken as the cut-off value. The contents of T cells, B cells and NK cells in patients from the two groups were measured. The results showed that HO1 expression was not associated with CD4+ T, B, CD8+ T cells in patients with AML, whereas the number of NK cells in patients with AML with high HO-1 expression was significantly lower than that in patients with AML with low HO-1 expression. (P < 0.01, Figure 1A,B). In addition, cytokines in patients were measured, and we found that the expression of interleukin-2 and interferon-γ in patients with high HO-1 expression was lower than that in low HO-1 expression group (P < 0.01, Figure 1C,D). To explore the role of HO-1 in patients with AML, HO-1 was detected by western blot in patients with different stages of AML. As a result, overexpression of HO-1 was found in relapsed patients (P < 0.01, Figure 1E,F). The expression of HO-1 was greater in the relapsed state than in the newly diagnosed state of the same patient (P < 0.01, Figure 1G,H). Therefore, we tentatively conclude that the aforementioned results suggest that HO-1 may inhibit NK cell cytotoxicity and lead to the immune escape of AML cells, causing a greater risk of relapse.
Fig. 1HO-1 expression is associated with weakened NK cells and a high recurrence rate in patients with AML. (A, B) Percentage of T, B and NK cells in patients with different HO-1 expression. *P < 0.05; ns, no significance. (C, D) Levels of interkeukin-2 (IL-2) and interferon-γ (IFN-γ) in patients with different HO-1 expression. **P < 0.01. (E, F) Protein expression of HO-1 in newly diagnosed and relapsed patients with AML was measured by western blot. **P < 0.01. (G, H) The expression of HO-1 was detected by western blot in the same patient at the initial diagnosis and relapse stage. **P<0.01. (*P < 0.05, **P < 0.01; ns, no significance).
Decreased HLA-C expression was associated with HO-1 overexpression in patients with AML
MHC Ⅰ molecules play an essential role in the cytotoxic process of NK cells. To further understand the specific mechanism of AML immune escape caused by HO-1, this work investigated the correlation between HO-1 and MHC Ⅰ molecule by protein–protein interaction network analysis. The results showed that HO-1 might affect HLA-C through physical linkage, and it might interact with HLA-DBQ1 through co-expression (Figure 2A,B). qRT-PCR was adopted for comparing HLA-C and HLA-DQB1 in patients with AML with different HO-1 expression levels. As a result, HLA-C was associated with the expression of HO-1 but not HLA-DQB1 at the mRNA level (P < 0.05, Figure 2C,D). Therefore, we will further investigate the relationship between HLA-C and HO-1 in subsequent studies. To evaluate the relation of HO-1 with HLA-C expression, this work assessed HO-1 and HLA-C expression levels in cells from 15 healthy donors and 30 patients with AML, including 15 newly diagnosed patients and 15 relapsed patients. The characteristics of patients with AML are shown in supplementary Table 1. It was found that HO-1 expression in patients with AML was greater than that in healthy donors (P < 0.05, Figure 2E). Comparatively, HLA-C expression was lower than that in healthy donors (P < 0.01, Figure 2F). In addition, the plasma HO-1 levels gradually increased. By contrast, HLA-C expression levels gradually decreased from healthy donors to patients with relapsed AML. Pearman correlation analysis indicated that HLA-C expression was significantly correlated with HO-1 expression (r = 0.635, P < 0.001, Figure 2G). Western blot was used to detect the expression of HLA-C in patients with AML with different HO-1 expression. According to our results, the expression of HLA-C was lower in patients with AML with high HO-1 expression (P < 0.001, Figure 2H-J). The ICC analysis as performed to detect HLA-C. The ICC staining results suggested that patients in the HO-1 overexpression group had a low HLA-C expression level (Figure 2K). These results indicated that HO-1 expression was inversely correlated with HLA-C in patients with AML.
Fig. 2HLA-C expression is associated with decreased HO-1 expression in patients with AML. (A, B) Protein–protein interaction network analysis of the interaction between HO-1 and MHC I/II antigens showed that HO-1 could affect HLA-C through physical linkage. HO-1 can interact with HLA-DBQ1 through co-expression. (C, D) The expression of HLA-C and HLA-DQB1 mRNA in patients with AML with different HO-1 expression levels was detected by qRT-PCR. β-actin was used as a control. *P < 0.05; ns, no significance. (E, F) mRNA expression of HO-1 and HLA-C in primary human cells were measured by qRT-PCR. β-actin was used as a control. *P < 0.05, **P < 0.01 versus normal group. (G) A Pearman correlation analysis showed the association between HO-1 mRNA expression level and HLA-C mRNA level (r = 0.612; P < 0.001). (H-J) Western blot measured protein expression of HO-1 and HLA-C in patients with AML. * P < 0.05, *** P < 0.001. (K, L) Representative images of ICC staining of HLA-C in patients with AML (P1, HO-1-low group; P5, HO-1-high group), Scale bars: 100 and 50 μm from left to right (*P < 0.05, **P < 0.01, ***P < 0.001; ns, no significance).
Up-regulation of HO-1 significantly inhibited HLA-C expression in AML cells
Next, this work determined whether HO-1 regulated HLA-C expression in AML cells. First, western blot was adopted for detecting four AML cell lines. The results demonstrated that among these four AML cell lines, the U937 cell line had the greatest HO-1 expression, whereas the THP-1 cell line had the lowest HO-1 expression (see supplementary Figure 1A). Therefore, THP-1 cells were treated with hemin to induce HO-1 expression. Western blot and qRT-PCR assays demonstrated that with the increases in hemin concentration (from 25 to 50 μmol), the expression of HLA-C gradually decreased in THP-1 cells (P < 0.05, Figure 3A-E). At the same time, the HO-1–overexpressed AML cell line THP-1-HO-1 and its control were established by lentiviruses. Western blot and qRT-PCR were adopted to confirm the transfection efficiency. Western blot, qRT-PCR and flow cytometry showed that overexpression of HO-1 decreased HLA-C expression (P < 0.05, Figure 3F-K). Then, HO-1 expression was inhibited with pharmacological inhibitor ZnPPIX in U937 cells. The results demonstrated that with the increase in ZnPPIX concentration (from 25 to 50 μmol), HLA-C expression gradually increased in U937 cells (P < 0.05; Figure 3L-P). Thereafter, HO-1–silenced AML cell line U937-Si-HO-1 and its control were established by lentiviruses. Western blot, qRT-PCR and flow cytometry revealed that HLA-C was overexpressed after HO-1 silencing. (Figure 3Q-V) These results suggest that HO-1 may inhibit HLA-C expression in AML cells.
Fig. 3HO-1 inhibited HLA-C expression in AML cell lines. (A-C) Western blot analysis of HO-1 and HLA-C protein levels of THP-1 cells after hemin treatment for 24 h. β-actin was used as the load control. (D, E) The expression of HO-1 and HLA-C mRNA in THP-1 cells was detected by qRT-PCR. β-actin was used as a control. Cells were treated with hemin for 24 h. *P < 0.05 versus untreated control group (0 μmol); #P < 0.05, ##P < 0.01 versus low hemin group (=25 μmol). (F-H) After transfection of THP-1 cells with EV and HO-1 recombinant lentivirus, the protein expressions of HO-1 and HLA-C in HO-1 overexpressed THP-1 cells were detected by western blot. *P < 0.05 versus THP-1-EV group. (I, J) The mRNA levels of HO-1 and HLA-C were detected by qRT-PCR. **P < 0.01 versus THP-1-EV group. (K) The expression of HLA-C in EV THP-1 and THP-1-HO-1 cell lines was analyzed by flow cytometry. (L-N) The levels of HO-1 and HLA-C proteins were analyzed by western blot after ZNPP treatment for 24 h. (O, P) The mRNA expressions of HO-1 and HLA-C in U937 cells were detected by qRT-PCR. *P < 0.05 versus the untreated control group (0 μmol); #P < 0.05 versus the low ZNPP group (=25 μmol). (Q-S) After transfection of U937 cells with EV and HO-1 silencing recombinant lentivirus, the protein expressions of HO-1 and HLA-C in HO-1 silencing U937 cells were detected by western blot. (T, U) The mRNA levels of HO-1 and HLA-C were detected by qRT-PCR after transfection with EV and HO-1 silencing recombinant lentivirus. *P < 0.05 versus U937-EV group. (V) The expression of HLA-C in EV U937 and U937-Si-HO-1 cell lines was analyzed by flow cytometry (*P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, ###P < 0.001).
Silencing HO-1 expression in AML cells up-regulated HLA-C expression and enhanced immune escape in vivo
To confirm the effect of HO-1 on the growth of AML cells in vivo, mice were divided into four groups: EV, Si-HO-1, EV + NK cells and Si-HO-1+ NK cells, with three mice in each group. AML xenograft mouse models were established by subcutaneously injecting U937 cells transfected with silenced HO-1 or empty vector. NK cells were administered to the mice via the tail vein immediately after the tumor became palpable. The results revealed that HO-1 silencing resulted in a significant increase in tumor growth in comparison with the EV group (Figure 4A). Compared with the EV group, HO-1 silencing significantly inhibited tumor weight (P < 0.05, Figure 4C) and tumor volume (P < 0.01, Figure 4D). In addition, NK-cell injection significantly reduced tumor weight (P < 0.01, Figure 4C) and tumor volume (P < 0.001, Figure 4D). The survival time of mice in the HO-1 silencing group was greater than that in the EV group. The survival time of mice in NK cell injection group was greater than that in non-injection group (P < 0.05, as shown in Figure 4B). In the case of NK-cell injection, the tumor volume (P < 0.001, Figure 4D) and tumor weight (P < 0.01, Figure 4C) of the HO-1 silencing group were significantly lower than those of the EV group, and the survival time was greater than that of the EV group (P < 0.05, Figure 4B). Immunohistochemistry was employed to detect the expression of HLA-C in paraffin-embedded tumor tissues. In vivo, HLA-C expression was greater in the HO-1 silencing group than in the EV group (P < 0.01, Figure 4E-H). Thus, these data suggest that HO-1 silencing inhibits tumor growth and promotes HLA-C expression while facilitating NK-cell cytotoxicity in mice.
Fig. 4AML cells with HO-1 silencing expression had a lower risk of immune escape in vivo. (A) Images of subcutaneous xenografts from mice in the EV, Si-HO-1, EV1 + NK and Si-HO-1 + NK groups. n = 12. (B) Survival analysis curves for subcutaneous xenografts. Survival was plotted by using the Kaplan–Meier method. (C) Tumor weight change curves for subcutaneous xenografts. (D) Tumor volume growth curves for subcutaneous xenografts. (E-G) The expression of HO-1 and HLA-C was examined in xenograft tumor tissue sections using immunohistochemistry. *P < 0.05, **P < 0.01.
Inhibition of HO-1 enhanced the cytotoxicity of NK cells against AML cells by overexpression of HLA-C in AML cells
On the basis of the aforementioned clinical sample results, it was speculated that inhibition of HO-1 led to HLA-C overexpression and enhanced the cytotoxicity of NK cells to AML cells. To test this hypothesis, lentiviruses were used to construct AML cell lines (THP-1 and U937) with HO-1 overexpression and silencing. PBMCs were obtained from different healthy donors, and NK cell contents in PBMCs was determined by flow cytometry (see supplementary Fig. 2) Thereafter, the constructed AML cells were co-cultured with PBMCs, and the NK cells were sorted by flow cytometry to detect the changes in CD107a. The results showed that in AML cell lines, HO-1 silencing enhanced the cytotoxicity of NK cells to AML cells (P < 0.01, Figure 5A,B), whereas HO-1 overexpression weakened the cytotoxicity of NK cells to AML cells (P < 0.05, Figure 5C, D). In contrast, the LDH assay was used to verify the effect of HO-1 on cytotoxicity of NK cells in AML cells. The results demonstrated that down-regulation of HO-1 significantly increased cytotoxicity of NK cells, whereas up-regulation of HO-1 significantly decreased cytotoxicity of NK cells to AML cells (Figure 5I,J)
Fig. 5HO-1 attenuated the cytotoxicity of NK cells to AML cells by inhibiting HLA-C expression. (A, B) U937 cells were transfected with EV, HO-1–silenced recombinant lentivirus and then co-cultured with PBMCs from healthy donors. CD107a expression of NK cells was detected by flow cytometry. (C, D) THP-1 cells transfected with EV and HO-1 recombinant lentivirus were then co-cultured with PBMCs from healthy donors. CD107a expression of NK cells was detected by flow cytometry. (E-H) U937-Si-HO-1 and THP-1 cells were transfected with HLA-C silenced small interfering RNA, then the treated group and untreated group were co-cultured with PBMCs from healthy donors. CD107a expression of NK cells was detected by flow cytometry. (I, J) LDH assay was used to verify the effect of HO-1 on cytotoxicity of NK cells in THP-1 and U937 cell lines. (K, L) The effect of HLA-C on cytotoxicity of NK cells in THP-1 and U937 cell lines was verified by LDH assay. (*P < 0.05; **P < 0.01).
To confirm the effect of HLA-C on NK cell toxicity, HLA-C expression was silenced with small interfering RNA in THP-1 and Si HO-1 U937 cell lines and established a control group. Western blot was used to verify the transfection efficiency (see supplementary Figure 1B-E). The silenced cells were co-cultured with PBMCs, and the expression of CD107a in NK cells was detected by flow cytometry. As a result, the expression of CD107a in NK cells after HLA-C silencing was lower than control groups of U937 and THP-1 cell lines (P < 0.05; Figure 5E-H). In contrast, LDH assay was used to detect the effect of HLA-C on the cytotoxicity of NK cells to AML cells, and the results showed that the cytotoxicity of NK cells to AML cells decreased after HLA-C expression was silenced (Figure 5K,L). Those results indicated that HLA-C silencing inhibited the cytotoxicity of NK cells to AML, and HO-1 might affect the cytotoxicity of NK cells via HLA-C. Our previous experimental results demonstrated that HO-1 overexpression silenced HLA-C. At the same time, HLA-C silencing inhibited the cytotoxicity of NK cells to AML cells, suggesting that HO-1 affected NK cell cytotoxicity via HLA-C.
HO-1 downregulates HLA-C expression by activating the JNK/c-Jun axis in AML cells
Previous experiments in our group demonstrated that HO-1 protected AML by activating the JNK/c-Jun signaling pathway [
] showed that activation of the JNK/c-Jun signaling pathway inhibited the cytotoxicity of NK cells. The aforementioned studies suggest that the JNK/c-Jun activation plays an essential role in AML and NK-cell cytotoxicity. The results revealed that HO-1 might regulate HLA-C through the JNK/c-Jun signaling pathway. Therefore, western blot assay was conducted to confirm this result. Our results indicated that the phosphorylated levels of JNK and C-Jun in THP-1 cells significantly increased through up-regulating HO-1 expression (P < 0.05; Figure 6A,B), whereas silencing HO-1 expression reduced the phosphorylation levels of JNK and c-Jun levels in U937 cell lines (P < 0.05; Figure 6C,D). Based on the obtained results, HO-1 was positively correlated with the JNK/c-Jun axis. In addition, the JNK inhibitor (SP600125) was selected to complete the following experiment. Subsequently, after treatment with 10 μmol of SP600125 for 24 h, HO-1 expression in THP-1 and U937 cells was slightly changed, whereas the protein levels of p-JNK and p-c-Jun decreased. Conversely, the protein levels of HLA-C were elevated (Figure 6A-D). Collectively, these data suggest that HO-1 overexpression inhibits HLA-C expression by activating the JNK/c-Jun signaling pathway.
Fig. 6HO-1 inhibits HLA-C through the JNK/c-Jun signaling pathway. (A, B) THP-1 cells were treated with 10 μmol SP600125 for 24 h, and the protein expression levels of HO-1, HLA-C, JNK, pJNK, c-Jun and p-c-Jun in the HO-1 overexpressed group, EV group and untreated group were detected by western blot. The relative gray values are displayed in the form of a histogram. (C, D) U937 cells were treated with 10 μmol SP600125 for 24 h, and the protein expression levels of HO-1, HLA-C, JNK, pJNK, c-Jun and p-c-Jun in the HO-1 silenced, and EV groups and untreated groups were detected by western blot. The relative gray values are displayed in the form of a histogram. Data are presented as mean ± standard deviation of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).
This study revealed the biological and immunological role of HO-1 in AML. In this study, HO-1 overexpression led to HLA-C deletion and attenuated the cytotoxicity of NK cells to AML cells. According to our results, mice with HO-1–silenced AML cells exhibited lower leukemic infiltration, greater NK cytotoxicity, greater survival and HLA-C overexpression in vivo. In AML cells, HO-1 overexpression activated the JNK/c-Jun pathway to inhibit HLA-C expression, thereby inhibiting NK-cell cytotoxicity to AML cells (Figure 7). The HO-1 levels elevated in patients with relapsed AML, and the prognosis was generally poor. Therefore, targeting the HO-1/JNK/c-Jun/HLA-C axis may be a promising approach for the successful treatment of AML.
Fig. 7Mechanism of HO-1–mediated immune escape in AML cells. HO-1 positively regulates JNK, activates phosphorylated c-Jun, leads to HLA-C inhibition and ultimately inhibits NK cell cytotoxicity to AML cells, resulting in immune escape.
Although there are many treatments for AML, the cure rate remains unsatisfactory. Therefore, it is essential to identify potential therapeutic targets for AML. HO-1 has been found to play an important role in cancer cell proliferation and drug resistance [
] reported that down-regulation of HO-1 might contribute to the sensitivity of melanoma to NK-cell–mediated recognition and killing. Our group found that HO-1 inhibited the cytotoxicity of NK cells to AML cells in previous experiments [
]. Our current study was consistent with these observations. This work isolated and compared HO-1 expression in cells from healthy donors and patients with AML at different stages. HO-1 expression was greater in patients with relapsed AML than in newly diagnosed AML. Furthermore, inhibition of HO-1 increased the cytotoxicity of NK cells against AML cells. More importantly, the effect of HO-1 on NK cell cytotoxicity in AML by affecting HLA-C has not been reported. However, the underlying mechanism of HO-1 involvement in the immune escape of AML cells needs to be further investigated.
The immune escape of cancer cells is one of the main difficulties in cancer therapy. Previous studies have concluded that the immune escape of AML is associated with NK-cell cytotoxicity [
] found that reduced expression of HLA-C in leukemia was associated with a greater rate of relapse. Our results were consistent with previous studies. According to our results, HLA-C silencing weakened the toxicity of NK cells to AML cells, leading to AML relapse. Interestingly, HLA-C expression level was negatively related to HO-1 expression level in patients with AML, implying a potential association between HO-1 and HLA-C. No evidence has been found on the relationship between HO-1 and HLA-C in the occurrence of AML immune escape.
Understanding the mechanism by which HO-1 inhibits HLA-C in AML will significantly facilitate the development of AML-related therapies. Continuous activation of HO-1 leads to a relative decrease in intracellular HLA-C. In addition, specific concentrations of HLA-C can promote the cytotoxicity of NK cells to AML cells. Previous studies by our group [
] showed that JNK/c-Jun activation played a vital role in AML recurrence and NK-cell cytotoxicity. Our study found that HO-1 inhibited HLA-C expression and reduced NK cytotoxicity in AML cells by activating the JNK/c-Jun signaling pathway. Moreover, this provides a new understanding of promoting immune escape of AML cells.
In conclusion, our work describes the changes in NK-cell function induced by the HO-1/HLA-C axis in AML, further deepening our current understanding of the effect of HO-1 on NK-cell function in AML. NK-cell–mediated innate immunity is important in the fight against tumors, especially when acquired immunity is depleted and dysfunctional [
]. Therefore, anti-HO-1 therapy, which can promote the anti-tumor effect of NK cells, may play an important role in combating AML.
Declaration of Competing Interest
The authors have no commercial, proprietary or financial interest in the products or companies described in this article.
Funding
This research was funded by the National Natural Science Foundation of China (grant no. 82170168), National Clinical Research Center for Hematological Diseases (The First Affiliated Hospital of Soochow University, no. 2020ZKPB03), Beijing Bethune Foundation Committee (no. B19153DT) and National Natural Science Foundation General Fund Cultivation Program of Affiliated Hospital of Guizhou Medical University (no. Gyfynsfc-2021-3) to JW. The experiments conducted in this study comply with the current laws of China.
Author Contributions
Conception and design of the study: C.F, T.Z, J.W. Acquisition of data: C.F, T.Z, L.W, Q.K. Analysis and interpretation of data: C.F, C.P, X.L, Q.S. Drafting or revising the manuscript: C.F, S.C, T.H. All authors have approved the final article.
Acknowledgments
We thank our colleagues in the laboratory for helpful discussions.
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Natural killer cells in myeloid malignancies: Immune surveillance, NK cell dysfunction, and pharmacological opportunities to bolster the endogenous NK cells.
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